A Drosophila Model for Angelman Syndrome

天使综合症的果蝇模型

• 批准号: 7026987
• 负责人: JANICE A. FISCHER
• 金额: $ 16.94万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7026987
• 关键词: Drosophilidae; acid aminoacid ligase; developmental genetics; disease /disorder model; enzyme substrate; gene expression; genetic models; genetic screening; genetically modified animals; happy puppet syndrome; in situ hybridization; model design /development; mutant; phenotype; ubiquitin

DESCRIPTION (provided by applicant): Angelman syndrome is a neurological disorder affecting ~1/15,000 people that results in severe mental retardation. The disease is caused by loss-of-function mutations in Ube3a, which encodes a HECT-domain ubiquitin ligase or E3 protein. E3 proteins are specificity factors for ubiquitination; they bind specific substrates and bring the ubiquitination machinery to them. A simple model for the biochemistry of Angelman syndrome is that loss of UbeSa activity results in overexpression of the E3's normal substrates. The Drosophila genome contains a Ube3a homolog called CG6190. The goal of the research proposed is to generate a Drosophila model for Angelman syndrome that will allow a test of this hypothesis and the identification of the substrates relevant to the disease. First, it will be determined if Drosophila CG6190 knock-out mutants have an Angelman syndrome-like phenotype. Mutant flies will be analyzed for morphological and functional defects in their central nervous system. Second, the expression pattern of CG6190 mRNA and protein during Drosophila development will be described. In situ hybridization will be used to detect mRNA in whole organisms and anti-CG6190 antibodies will be generated and used to detect the protein. Third, it will be determined if Drosophila CG6190 and human UbeSa are functional homologs. By generating transgenes, the abilities of wild-type CG6190 and human UbeSa to complement the CG6190 mutant phenotype and to generate mutant phenotypes when overexpressed will be compared. In addition, site-directed mutagenesis will be used to generate alleles of the Drosophila and human genes that contain mutations identified in Angelman syndrome patients. The effects of expressing the resulting mutant alleles will be tested similarly. Finally, a genetic screen will be performed in Drosophila to identify candidate CG6190/Ube3a substrates. Overexpression of wild-type CG6190 in the Drosophila eye results in a severe mutant phenotype. Modifiers of the eye phenotype will be isolated and the mutant genes identified.

描述（由申请人提供）：Angelman综合征是一种神经系统疾病，影响约1/15，000人，导致严重精神发育迟滞。这种疾病是由Ube 3a的功能缺失突变引起的，Ube 3a编码HECT结构域泛素连接酶或E3蛋白。E3蛋白是泛素化的特异性因子，它们结合特定的底物并将泛素化机制带到底物处。Angelman综合征生物化学的一个简单模型是UbeSa活性的丧失导致E3正常底物的过度表达。果蝇的基因组中含有一个叫做CG 6190的Ube 3a同源物。这项研究的目的是建立一个安格尔曼综合征的果蝇模型，以检验这一假设，并确定与该疾病相关的底物。首先，将确定果蝇CG 6190敲除突变体是否具有Angelman综合征样表型。突变果蝇将被分析其中枢神经系统的形态和功能缺陷。其次，将描述果蝇发育过程中CG 6190 mRNA和蛋白质的表达模式。原位杂交将用于检测整个生物体中的mRNA，并将产生抗CG 6190抗体并用于检测蛋白质。第三，将确定果蝇CG 6190和人UbeSa是否是功能同源物。通过产生转基因，将比较野生型CG 6190和人UbeSa补充CG 6190突变体表型和在过表达时产生突变体表型的能力。此外，定点诱变将用于产生含有在Angelman综合征患者中鉴定的突变的果蝇和人类基因的等位基因。将类似地测试表达所得突变等位基因的影响。最后，将在果蝇中进行遗传筛选以鉴定候选CG 6190/Ube 3a底物。野生型CG 6190在果蝇眼睛中的过表达导致严重的突变表型。将分离眼睛表型的修饰物并鉴定突变基因。